US5639599A - Amplification of nucleic acids from mononuclear cells using iron complexing and other agents - Google Patents

Amplification of nucleic acids from mononuclear cells using iron complexing and other agents Download PDF

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US5639599A
US5639599A US08/254,310 US25431094A US5639599A US 5639599 A US5639599 A US 5639599A US 25431094 A US25431094 A US 25431094A US 5639599 A US5639599 A US 5639599A
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complexing agent
nucleic acid
kit
band
agent
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Thomas B. Ryder
Daniel L. Kacian
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Gen Probe Inc
Cytyc Corp
Third Wave Technologies Inc
Hologic Inc
Suros Surgical Systems Inc
Biolucent LLC
Cytyc Surgical Products LLC
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides

Definitions

  • the field of the present invention is the preparation of nucleic acid for study, research and investigation.
  • nucleic acid it is common to require nucleic acid to be isolated and purified (i.e., prepared) from various tissues in order to detect the presence of a particular nucleic acid--for example, the presence of HIV-1 DNA or RNA in a blood cell of a human.
  • nucleic acid is generally extracted after extensive purification of appropriate blood cells, lysis of these cells and purification of the released nucleic acids to remove substances that might inhibit later analytical procedures.
  • RNA polymerase chain reaction Two common methods which allow amplification of a specified sequence of nucleic acid (e.g., deoxyribonucleic acid (DNA) or ribonucleic acid (RNA)) are one termed the "polymerase chain reaction" (where two primers are used to synthesize nucleic acid lying between the regions where the primers hybridize), and one which uses RNAse H, reverse transcriptase and RNA polymerase.
  • the present invention is directed to methods and kits for the preparation of nucleic acid, and particularly for isolation of DNA or RNA from cells, such as mononuclear cells (e.g., T-lymphocytes and/or monocytes), for amplification of that nucleic acid.
  • Such amplified nucleic acid may then be used for various purposes, including screening the nucleic acid for the presence of viral nucleic acid sequences, using a probe which is complementary to a selected nucleic acid sequence of the virus. It is also useful for detection of genetic anomalies or defects in the nucleic acid. Accordingly, the methods and kits are designed to allow rapid and easy preparation of nucleic acid without the need for extensive purification procedures.
  • the present invention features a method for preparing nucleic acid from cells for amplification.
  • a sample containing various cells e.g., whole blood
  • an appropriate centrifugation medium are centrifuged to cause a population of one cell type to gather in a discrete layer.
  • This layer is separate and distinct from the remainder of the cells and detritus in the sample, except for a small amount of platelets and/or lipids or other low density components, or other soluble and suspended constituents.
  • the presence of platelets and other components does not prevent amplification of nucleic acid purified in this method.
  • the population of cells includes mononuclear cells from whole blood.
  • the cells may be cells other than mononuclear cells and/or may be derived from other sources, such as pleural fluid, synovial fluid, or an in vitro source of cells.
  • the cells or sample of cells need only be available to be centrifuged for separation, or in a state to be lysed (as discussed below), and then effectively used in later reactions according to the present invention.
  • the centrifugation medium may be isotonic and/or isopycnic to the cells, and is preferably of a density intermediate between that of the desired cells and of the other cells in the sample.
  • the cells are removed from the centrifugation composition, for example by suction using a pipette, and lysed. Such lysis makes the nucleic acid associated with the cells available for the amplification.
  • the method includes adding a complexing agent that complexes with, and effectively removes, ferric ions (Fe +++ ) from the solution before amplifying the nucleic acid.
  • the presence of ferric ions interferes with certain enzymatic procedures, such as those used for nucleic acid amplification.
  • the complexing agent is preferably selected so that it has greater affinity, e.g., at least four logs greater, for ferric ions than for (Zn ++ ), manganese ions (Mn ++ ) or magnesium ions (Mg ++ ), as these ions are beneficial and tend to facilitate these enzymatic reactions.
  • Such an agent is the chelating agent deferoxamine (also known as desferrioxamine and desferrioxamine B).
  • a second or different complexing agent may be added that complexes with calcium ions (Ca ++ ), or other ions that interfere with an amplification reaction or other reactions performed with the lysate of the present invention. Calcium ions may also interfere with enzymatic reactions used in amplification procedures.
  • the second complexing agent is most preferably selected so as to fail to complex with zinc ions or magnesium ions, or to have more affinity for undesirable ion species (such as Fe +++ and Ca ++ ) than for desirable ion species (such as Zn ++ , Mn ++ , and Mg ++ ).
  • zinc ions, manganese ions or magnesium ions may be added to the solution to facilitate enzymatic reactions.
  • the invention features a method of amplifying nucleic acid in which the nucleic acid to be amplified is produced in a solution with ferric ions, and those ferric ions effectively removed from the mixture by use of a complexing agent such that amplification can occur.
  • the solution may also contain calcium ions, which are preferably also removed from the solution by a complexing agent, so that amplification may occur.
  • the invention features a method for amplifying nucleic acid from whole cells.
  • the whole cells are lysed prior to amplification by the use of a strong alkali such as KOH, NaOH or LiOH.
  • a strong alkali such as KOH, NaOH or LiOH.
  • Such a lysis also denatures any double stranded nucleic acid from the cells, thereby facilitating amplification.
  • a ferric ion complexing agent is preferably added to remove ferric ion from the suspension or composition.
  • nucleic acid hybridization and detection techniques are known in the art for screening the amplified nucleic acid using known nucleic acid hybridization and detection techniques. For example, by using a nucleic acid probe complementary to a target sequence (which preferably includes the sequence targeted for amplification), hybridizing the probe to the target nucleic acid sequence, and detecting the complex of probe and target by well known methods, such as Southern or northern blots or by the homogeneous solution phase procedure ("HPA") described in Arnold et al, Clin. Chem., 35: 1588 (1989), and PCT U.S. Ser. No. 88/02746, all of which are hereby incorporated by reference.
  • HPA homogeneous solution phase procedure
  • a probe detection method it can be determined whether vital nucleic acid, such as that from HIV or hepatitis B virus (HBV), is associated with the nucleic acid present in the sample.
  • vital nucleic acid such as that from HIV or hepatitis B virus (HBV)
  • HBV hepatitis B virus
  • Such a screening may also target any other sort of nucleic acid sequence, such as a genetic anomaly or defect.
  • the use of such amplification procedures allows even a single copy of the targeted nucleic acid in the portion of the sample recovered after centrifugation to be detected using the present invention.
  • the kit may include a supply of the centrifugation medium (and an osmotic agent, if necessary), a supply of a lysing agent sufficient to lyse the cells from the sample, and a supply of the amplification materials sufficient to amplify the nucleic acid from the cells.
  • a kit may also include a ferric ion and/or calcium ion complexing agent, and, a supply of the probe for a desired nucleic acid target, or a known genetic anomaly.
  • FIG. 1 is a graph depicting the red blood cell counts, hematocrits, and plasma density for 66 different individuals.
  • FIG. 2 is a chart depicting the effects on amplification of zinc titration in concert with deferoxamine.
  • FIG. 3 is a chart depicting the effects of polyethyleneimine (PEI) and deferoxamine on amplification.
  • the claimed method features a series of steps for the collection, isolation, preparation, amplification and screening of nucleic acid, preferably from mononuclear cells, and a combination of apparatus, media and agents to effectuate such a method.
  • the various steps, apparatus, media and agents are discussed generally above, and examples are now provided.
  • a sample of cells may be provided from any suitable source, such as from whole blood, or from synovial fluid, pleural fluid, or other fluids containing cells of interest.
  • the sample comprises mononuclear cells in anticoagulant treated whole blood, which may be obtained from any animal, including a human being.
  • the blood may be pretreated if desired, e.g., to remove red blood cells and/or fibrin, provided that the mononuclear cells (or other cells of interest) remain in the fluid.
  • Other blood fractions or any other fluids containing cells of interest can be used.
  • the blood, or other source of a sample of cells may be obtained using any available method, including those known in the art.
  • the sample can be obtained and stored, and even subjected to centrifugation, in the same device.
  • an anti-coagulant such as EDTA or Heparin, so that the blood does not coagulate prior to performance of the rest of the method, or during use of the rest of the materials and apparatus in the kit.
  • centrifugation media are known in the art.
  • the centrifugation medium may be isotonic to the cells of the sample, and may also be generally isopycnic to the cells from the sample or, preferably, of a density intermediate the cell population of interest and other cell populations in the sample.
  • the centrifugation medium and the sample of cells may be mixed prior to centrifugation such that there is no density gradient prior to centrifugation.
  • isopycnic it is meant that the centrifugation medium is approximately the same density as the cells in the sample, such that, upon centrifugation, desired cells from the sample will separate from the remainder of the mixture, which remainder, in the case of a sample from whole blood, will contain red blood cells and other white blood cells.
  • the composition can be formed as a density gradient prior to or during centrifugation.
  • the sample of cells is mixed with a centrifugation medium such that the resulting density is intermediate between the buoyant density of the cell population of interest and other cell populations in the sample.
  • the density of the mixture is greater than the buoyant density of mononuclear cells and less than the buoyant density of red blood cells and granulocytes in a sample of whole blood.
  • the centrifugation mixture is subjected to centrifugation such that the mononuclear cells "float" to the meniscus in a relatively narrow band. Centrifugation of such a mixture for longer than such time, for example until isopycnic equilibrium is reached, will result in a broader band (or bands) of desired cells.
  • the final desired density of the centrifugation mixture is about 1.077 grams per milliliter of fluid to separate mononuclear cells.
  • the density of the centrifugation medium and the mixture will vary according to the osmolarity, type and volume of the cells in the sample.
  • isotonic it is meant that the mixture, once it is made, is approximately isotonic to the cells from the sample, such that the cells do not rupture pursuant to the effects of an osmotic difference.
  • this isotonic state is achieved through the use of an osmotic agent, such as sucrose or an appropriate salt.
  • An appropriate salt is a salt that does not otherwise interfere with the performance of the method; accordingly, a salt which effectively prohibits the amplification of the nucleic acids is not desirable. It is sometimes advantageous if the osmotic agent does not add to the ionic concentration in the mixture, e.g., such as when the osmotic agent is sucrose.
  • the centrifugation medium preferably is able to be used directly in the sample, such as blood, and does not lyse the cells in the sample.
  • useful centrifugation media include PERCOLL, SEPRACELL-MN, and NYCO-DENZ.
  • Other useful media, including Ficoll-hypaque or Ficoll-isopaque, are typically used by layering rather than mixing. For example, if the centrifugation medium lyses white blood cells, then nucleic acid may be prematurely released. If red blood cells are lysed they release hemoglobin, and therefore a source of ferric ion, which interferes with the later amplification reactions. It is preferred that the centrifugation medium allow more than or equal to about 50% of mononuclear cells present in a sample of whole blood to be recovered after centrifugation.
  • the mixture is then subjected to centrifugation such that the desired cells effectively move to form a discrete band of isolated cells.
  • the desired cells effectively move toward the end of the centrifuge tube that is toward the center of rotation, rising to the top of the centrifugation medium to form an upper portion containing isolated cells, along with some platelets and/or lipids and other constituents.
  • the mixture is centrifuged at about 2,900 rpm for about 20 minutes (about 1,500 ⁇ g).
  • the centrifugation is gentle enough that the centrifugation does not cause a substantial part, or significant percentage, of the cells to lyse, which means that sufficiently few cells lyse that the amplification of nucleic acid recovered from intact cells may proceed.
  • the centrifugation may be performed in a centrifugation tube wherein the part of the tube near the discrete band (e.g., the part of the tube near the center of rotation in isopycnic centrifugation), is formed so as to accentuate that portion of the composition containing the desired cells, such as mononuclear cells. This may be done, for example, by narrowing the interior diameter of the tube.
  • the centrifugation may be performed using a device that will inhibit turbulence, and therefore mixing, while making the composition, while performing centrifugation, and/or while removing the isolated, desired cells.
  • An example of such a device is a porous nylon filter that will allow desired cells to pass through it.
  • the portion of fluid containing the desired cells can be removed from the rest of the mixture using any means known in the art, such as by suctioning the portion using a pipette.
  • any means known in the art such as by suctioning the portion using a pipette.
  • less than about 20% of the total original volume is removed, e.g., about 500 ul of 6 ml, and preferably only about the top 100 ul at or adjacent the meniscus.
  • the cells After removing the isolated desired cells, the cells are lysed to release the nucleic acid from, or associated with, the cells. This means any nucleic acid either attached to or found within or on the desired cells.
  • the cells may be lysed by any desired method, including, for example, the use of a strong alkali, use of an enzymatic agent, use of a detergent, use of osmotic shock, use of chaotropic concentrations of solutes, or use of sonic disruption.
  • the cells may be lysed by use of potassium hydroxide.
  • This alkali, or an equivalent alkali, e.g., NaOH or LiOH is particularly useful since it also inactivates cellular nucleases and thus prevents inhibition of later enzymatic reactions using the alkali-induced lysate, and may also denature double stranded nucleic acid.
  • An agent that complexes with ferric ion and/or calcium ion and/or other undesirable ions may be added to the density centrifugation medium or to the solution during or after lysis.
  • Complexes means that the agent effectively attaches itself to the ion, whether through chelation, coordination, covalent bonding or some other form of bonding or attachment, such that the ion may no longer prevent the later amplification reaction of the invention.
  • These ions are removed from the solution resulting from the lysis because they may inhibit enzymes that may be used in other steps of the method, such as amplifying the nucleic acid from mononuclear cells.
  • the complexing agent is a chelating agent that removes such ions from solution, but does not bind significantly with zinc ions nor magnesium ions, as these ions are helpful to some reagents that may be used in the later amplification reaction.
  • a chelating agent that removes such ions from solution, but does not bind significantly with zinc ions nor magnesium ions, as these ions are helpful to some reagents that may be used in the later amplification reaction. See generally Zinc in DNA Replication and Transcription, Wu, F. Y. H., and Wu, C. W., Ann. Rev. Nutr. 7:251 (1987).
  • chelating agents examples include deferoxamine and transferrin.
  • Deferoxamine has a 10 31 binding constant for the ferric ion, see Antioxidant Capacity of Desferrioxamine and Ferrioxamine in the Chemically-Initiated Lipid Peroxidation of Rat Erythrocyte ghost Membranes, Videla, C. A., et al., Biochem. Int'l, 16, 799 (May 1988), and binds preferentially to zinc and ferric ions magnesium ions.
  • Deferoxamine has a 10 11 .1 binding constant for Zn 2+ , and a 10 4 .3 binding constant for Mg 2+ .
  • Examples of calcium ion complexing agents are oxalate and citrate. Oxalate and citrate also bind Fe +++ with greater stability compared to Mg ++ , especially at neutral-alkaline pH. Other agents, such as EDTA, are not preferred chelating agents, as they bind zinc ion and magnesium ion, along with other divalent cations.
  • Some useful chelating agents may have the potential to bind desirable ions (e.g., Mg ++ , Zn ++ ), but may be beneficial if they bind undesirable ions preferentially.
  • desirable ions e.g., Mg ++ , Zn ++
  • EGTA binds Ca ++ in preference to Mg ++ .
  • concentration of EGTA is less than the concentration of Mg ++ , there will still be a desirable and predictable activity of free Mg ++ but trace levels of Ca ++ will be effectively sequestered.
  • a chelator is mixed with a desirable species, such as Mg ++ , prior to adding it to the reaction of interest, the free activity of Mg ++ contributed to the reaction from other sources will not be reduced by addition of the complexed chelator, but the chelator may effectively sequester preferentially bound species such as Ca ++ , especially if the association/dissociation rates are relatively rapid.
  • a chelating agent is particularly helpful when an anticoagulant has been used previously in the method, as the use of such an anticoagulant may cause or allow the release of ferric ions.
  • PEI polyethyleneimine
  • An effective amount of such a polycation is that amount necessary to reduce inhibition by a polyanion. It is surprising that a polycation such as PEI would have such a beneficial effect because nucleic acids themselves are densely charged anions.
  • the nucleic acid may now be amplified using any method known in the art, such as a polymerase chain reaction procedure, or a procedure described by Kacian, supra, using RNAse H, reverse transcriptase and RNA polymerase.
  • the amplification will target the same sequence of nucleic acid that will be the target of the probe, discussed below.
  • the nucleic acid amplification may be performed on the nucleic acid from the desired cells without first removing the debris from the centrifugation and lysis, nor otherwise "cleaning up" the portion containing the isolated desired cells, other than appropriately preparing the chemical balance of the solution to be subjected to amplification, for example, neutralizing the lysis agent such as potassium hydroxide.
  • the amplified nucleic acid is now ready to be screened for a desired sequence of nucleic acid using any procedure known in the art or other related procedures, such as hybridization using a strand of DNA or RNA complementary to the desired nucleic acid sequence, which complementary strand is known as a "probe.”
  • the probe may be hybridized to the nucleic acid and detected using any known detection technique, such as traditional Southern or Northern blotting techniques, or the homogeneous protection assay ("HPA") described by Arnold et al., supra.
  • the probe will detect the nucleic acid associated with an HIV virus.
  • a simple kit for performing the above method may be prepared from readily available materials and reagents.
  • the kit may be designed so that the reagents complement each other, for example, the osmotic agent may be sucrose and the centrifugation medium PERCOLL®.
  • PERCOLL® is provided by the manufacturer at a density in the range 1.130 ⁇ 0.005 g/ml. The precise density is supplied with each lot.
  • ⁇ Desired the volume (in ml) of PERCOLL® stock required can be estimated from the following equations: ##EQU1##
  • the density of 1.25M sucrose is 1.1607 g/ml.
  • the final term in the numerator of the first equation (1000-V Osmot -V PERCOLL ®) represents the volume of water added to bring the final volume up to one liter and assumes a nominal density of 1.000 g/ml for water.
  • the RI expected for the desired 1.110 g/ml suspension would thus be 1.3564.
  • PERCOLL® suspensions in other osmotica such as 0.15M NaCl.
  • the density of NaCl solutions is also available in the literature and can be calculated as a function of concentration.
  • 1.50M NaCl which can be used as a 10 ⁇ stock instead of the 5 ⁇ sucrose in the formulation above, has a density of 1.058 g/ml.
  • the density can be calculated from the RI of such a PERCOLL®/saline DCM 2 by the following equation:
  • a refractive index of 1.350 would correspond to the desired density of 1.110 g/ml for DCM 2 .
  • Agents other than PERCOLL® can be used to generate the desired density.
  • the desired properties of a density reagent include: sufficient solubility and specific gravity to yield solutions of at least the desired density, little osmotic activity of its own, and low viscosity in solution.
  • NYCODENZ® stock solutions can be made by dissolving it in water at a density greater than desired as the final DCM density (e.g. >0.35M).
  • the density of an aqueous NYCODENZ® solution can be predicted from its molarity:
  • the buoyant density of the cells present in the sample can change if the osmotic strength is changed.
  • the buoyant density of the cells decreases as the osmolarity is decreased and increases as the osmolarity is increased.
  • the near-physiological osmolarities described here are suited to the purpose of this method (preparing cells for subsequent amplification reactions).
  • Red Blood Cells (RBCs) are typically more sensitive to osmotic shock than White Blood Cells (WBCs) and the most suitable sample processing conditions are those which minimize the possibility of RBC lysis.
  • a density of 1.110 g/ml is preferred for the DCM to mix with whole blood in order to obtain a final mixture density of 1.077 g/ml to separate mononuclear cells from whole blood.
  • This density for the DCM was chosen empirically based on the results of several experiments including examinations of the number of cells recovered, of the purity of the desired fraction, i.e. low granulocyte contamination as determined by Wright staining, and by observing a typical ratio of lymphocytes-to-monocytes, as determined by Wright staining, among the cells recovered.
  • This DCM density is also a preferable density based on the typical buoyant density of mononuclear cells (usually considered to be ⁇ 1.077 g/ml) and the normal range of blood composition.
  • the packed volume of RBCs for most adult individuals is usually at least 35% of the total blood volume, although this can be lower for individuals with significant anemia.
  • FIG. 1 shows the RBC counts and hematocrits determined for blood taken from 66 different individuals.
  • the 2 nd y-axis shows the corresponding density of the DCM-plasma mixture which would result from mixing a specified volume of DCM with an equal volume of blood having the hematocrit directly opposite on the primary y-axis.
  • DCM/plasma mixture having a density between 1.076 g/ml-1.082 g/ml for the majority of individuals.
  • MNC mononuclear cell
  • mixtures throughout this density range should be very effective at separating MNC and granulocytes since the buoyant density of the granulocytes typically averages 1.086 g/ml and only a small fraction have a buoyant density ⁇ 1.082 g/ml.
  • nucleic acid target amplification and/or hybridization analysis even substantial granulocyte contamination is not a significant problem since these techniques have been designed to be specific for desired target sequences even in the presence of vast excess levels of non-target sequences.
  • MNC which include target cells for HIV infection
  • granulocytes which are not target cells for HIV infection
  • the invention does not require that the blood and DCM be combined in equal volumes or that the DCM density is constrained to be 1.110 g/ml. It is within the skill of the art without undue experimentation to calculate the appropriate volumes to combine for a wide range of DCM densities based on the partial volumes of the components and their respective densities as described above. Conversely, the appropriate DCM density to use can be calculated to accommodate the desired volume and ultimate mixture density specifications.
  • the invention may include a centrifugation procedure comprising at least one wash step.
  • a centrifugation procedure comprising at least one wash step.
  • the appearance of the MNC band will vary slightly depending on the specifics of the centrifugation and the centrifugation apparatus, such as the diameter of the tube, volume of blood used, etc., but will typically concentrate at the highest point of the meniscus.
  • the complete MNC band may be harvested by aspirating into a pipet, manipulating the pipet so that the tip remains in a region of dense cell accumulation. Continue until most of the MNC are recovered. Typically, this will result in collection of 300-700 ⁇ l of the MNC/DCM/plasma suspension.
  • lysis may also be used, for example, combinations of other basic solutions followed by neutralizing acid solutions, or enzymatic or mild detergent agents. Such methods, for mononuclear cells and for other cells, are known in the art.
  • the above lysate contains a crude mixture of biologically derived molecules, many of which are low molecular weight hydrolysis products of the original sample constituents.
  • the DNA present in these lysates while fragmented and denatured compared to its original structure within the cells, is still a suitable reactant for a variety of chemical and biochemical reactions, including as a template for nucleic acid target amplification.
  • the lysate is added directly to the amplification reaction as the source of the analyte, without further purification.
  • metal ions can inhibit enzyme activities through a variety of mechanisms, including displacement or substitution of requisite metal co-factors, or by forming stable complexes with susceptible moieties, including amino acid side chains which are necessary for catalytic activity or which result in deleterious steric changes in the enzyme or by promoting deleterious redox reactions.
  • Chelators are sometimes employed to sequester undesirable metals but it is not obvious in any particular case that an efficacious chelator can be identified which does not itself inhibit the enzyme activity, e.g., by complexing necessary co-factors or even stripping bound metals from the enzyme.
  • Deferoxamine a biologically-derived natural product with extremely high affinity for Fe(III), was found to be highly effective at neutralizing the inhibition imposed by lysates of blood fractions on the amplification reactions and was found to be well tolerated in the reaction over at least the concentration range of 0.001-1 mM.
  • An example of deferoxamine's use is described below and the beneficial effects demonstrated in FIGS. 2 and 3.
  • Zn(OAc) 2 One of the agents tested and found to be effective at counteracting the lysate sample inhibition of amplification was Zn(OAc) 2 .
  • reverse transcriptase has been reported to be a Zn metalloenzyme (as has T7 RNA polymerase, though this is no longer thought to be the case) it is not known if (or claimed that) the beneficial activity of Zn ++ in this context is related to the normal Zn ++ binding site of one or more enzymes in the reaction. It is possible that Zn ++ interacts with other site(s) on the enzyme(s) in a favorable way or that it interferes directly or indirectly with one of the inhibitors in reactions of this type. It was not obvious that conditions could be identified where net beneficial effects were observed.
  • PEI polyethyleneimine
  • Deferoxamine mesylate at 0-1 mM final concentration.
  • Zn(OAc) 2 at 0-0.1 mM final concentration.
  • PEI at 0-3 ⁇ 10 -5 % final concentration.
  • deferoxamine may also be used, as well as other zinc providers, such as ZnCl 2 or ZnSO 4 .
  • other polycationic polymers such as polyallyl amine or Polybrene® (hexadimethrine bromide) may also be used with or instead of PEI.

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DK0574227T3 (da) 2000-08-07
AU5207698A (en) 1998-04-23
ATE192196T1 (de) 2000-05-15
DE69328459D1 (de) 2000-05-31
EP0574227B1 (fr) 2000-04-26
ES2145030T3 (es) 2000-07-01
DE69328459T2 (de) 2000-09-07
AU706083B2 (en) 1999-06-10
AU713753B2 (en) 1999-12-09
WO1993025710A1 (fr) 1993-12-23
AU4530893A (en) 1994-01-04
EP0574227A2 (fr) 1993-12-15
CA2137563C (fr) 2005-06-28
EP0574227A3 (en) 1994-08-24
AU5207498A (en) 1998-03-12
AU687352B2 (en) 1998-02-26
JPH07507458A (ja) 1995-08-24
CA2137563A1 (fr) 1993-12-23
JP3494646B2 (ja) 2004-02-09

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